This invention relates to novel pharmaceutically useful compounds, in particular compounds that are, or are prodrugs of, competitive inhibitors of trypsin-like serine proteases, especially thrombin, their use as medicaments, pharmaceutical compositions containing them and synthetic routes to their production.
Blood coagulation is the key process involved in both haemostasis (i.e. the prevention of blood loss from a damaged vessel) and thrombosis (i.e. the formation of a blood clot in a blood vessel, sometimes leading to vessel obstruction).
Coagulation is the result of a complex series of enzymatic reactions. One of the ultimate steps in this series of reactions is the conversion of the proenzyme prothrombin to the active enzyme thrombin.
Thrombin is known to play a central role in coagulation. It activates platelets, leading to platelet aggregation, converts fibrinogen into fibrin monomers, which polymerise spontaneously into fibrin polymers, and activates factor XIII, which in turn crosslinks the polymers to form insoluble fibrin. Furthermore, thrombin activates factor V and factor VIII leading to a xe2x80x9cpositive feedbackxe2x80x9d generation of thrombin from prothrombin.
By inhibiting the aggregation of platelets and the formation and crosslinking of fibrin, effective inhibitors of thrombin would be expected to exhibit antithrombotic activity. In addition, antithrombotic activity would be expected to be enhanced by effective inhibition of the positive feedback mechanism.
Further, it is known that administration of prodrugs of thrombin inhibitors may give rise to improvements in:
(a) certain pharmacokinetic properties after administration of; and
(b) the prevalence of certain side effects associated with, those inhibitors.
The early development of low molecular weight inhibitors of thrombin has been described by Claesson in Blood Coagul. Fibrinol. (1994) 5, 411.
Blombxc3xa4ck et al (in J. Clin. Lab. Invest. 24, suppl. 107, 59, (1969)) reported thrombin inhibitors based on the amino acid sequence situated around the cleavage site for the fibrinogen Axcex1 chain. Of the amino acid sequences discussed, these authors suggested the tripeptide sequence Phe-Val-Arg (P9-P2-P1, hereinafter referred to as the P3-P2-P1 sequence) would be the most effective inhibitor.
Thrombin inhibitors based on dipeptidyl derivatives with an xcex1,xcfx89-aminoalkyl guanidine in the P1-position are known from U.S. Pat. No. 4,346,078 and International Patent Application WO 93/11152. Similar, structurally related, dipeptidyl derivatives have also been reported. For example International Patent Application WO 94/29336 discloses compounds with, for example, aminomethyl benzamidines, cyclic aminoalkyl amidines and cyclic aminoalkyl guanidines in the P1-position (International Patent Application WO 97/23499 discloses prodrugs of certain of these compounds); European Patent Application 0 648 780, discloses compounds with, for example, cyclic aminoalkyl guanidines in the P1-position.
Thrombin inhibitors based on peptidyl derivatives, also having cyclic aminoalkyl guanidines (e.g. either 3- or 4-aminomethyl-1-amidinopiperidine) in the P1-position are known from European Patent Applications 0 468 231, 0 559 046 and 0 641 779.
Thrombin inhibitors based on tripeptidyl derivatives with arginine aldehyde in the P1-position were first disclosed in European Patent Application 0 185 390.
More recently, arginine aldehyde-based peptidyl derivatives, modified in the P3-position, have been reported. For example, International Patent Application WO 93/18060 discloses hydroxy acids, European Patent Application 0 526 877 des-amino acids, and European Patent Application 0 542 525 O-methyl mandelic acids in the P3-position.
Inhibitors of serine proteases (e.g. thrombin) based on electrophilic ketones in the P1-position are also known. For example, European Patent Application 0 195 212 discloses peptidyl xcex1-keto esters land amides, European Patent Application 0 362 002 fluoroalkylamide ketones, European Patent Application 0 364 344 xcex1,xcex2,xcex4-triketocompounds, and European Patent Application 0 530 167 xcex1-alkoxy ketone derivatives of arginine in the P1-position.
Other, structurally different, inhibitors of trypsin-like serine proteases based on C-terminal boronic acid derivatives of arginine and isothiouronium analogues thereof are known from European Patent Application 0 293 881.
More recently, thrombin inhibitors based on peptidyl derivatives have been disclosed in European Patent Application 0 669 317 and International Patent Applications WO 95/35309, WO 95/23609, WO 96/03374, WO 96/25426, WO 96/31504, WO 97/02284, WO 97/46577, WO 96/32110, WO 98/06740, WO 97/49404 and WO 98/57932.
However, there remains a need for effective inhibitors of trypsin-like serine proteases, such as thrombin. There is a particular need for compounds which are both orally bioavailable and selective in inhibiting thrombin over other serine proteases. Compounds which exhibit competitive inhibitory activity towards thrombin would be expected to be especially useful as anticoagulants and therefore in the therapeutic treatment of thrombosis and related disorders.
According to the invention there is provided a compound of formula I, 
wherein
R1 represents H, C1-4 alkyl (optionally substituted by one or more substituents selected from cyano, halo, OH, C(O)OR1a or C(O)N(R1b)R1c) or OR1d;
R1d represents H, C(O)R11, SiR12R13R14 or C1-6alkyl, which latter group is optionally substituted or terminated by one or more substituent selected from OR15 or (CH2)qR16;
R12, R13 and R14 independently represent H, phenyl or C1-6 alkyl;
R16 represents C1-4 alkyl, phenyl, OH, C(O)OR17 or C(O)N(H)R18;
R18 represents H, C1-4 alkyl or CH2C(O)OR19;
R15 and R17 independently represent H, C1-4 alkyl or C1-3 alkylphenyl;
R1a, R1b, R1c, R11 and R19 independently represent H or C1-4 alkyl; and
q represents 0, 1 or 2;
Rx represents a structural fragment of formula IIa, IIb or IIc, 
xe2x80x83wherein
the dotted lines independently represent optional bonds;
A and E independently represent O or S, CH or CH2 (as appropriate), or N or N(R21) (as appropriate);
D represents xe2x80x94CH2xe2x80x94, O, S, N(R22), xe2x80x94(CH2)2xe2x80x94, xe2x80x94CHxe2x95x90CHxe2x80x94, xe2x80x94CH2N(R22)xe2x80x94, xe2x80x94N(R22)CH2xe2x80x94, xe2x80x94CHxe2x95x90Nxe2x80x94, xe2x80x94Nxe2x95x90CHxe2x80x94, xe2x80x94CH2Oxe2x80x94, xe2x80x94OCH2xe2x80x94, xe2x80x94CH2Sxe2x80x94 or xe2x80x94SCH2xe2x80x94;
X1 represents C2-4 alkylene; C2-3 alkylene interrupted by Z; xe2x80x94C(O)xe2x80x94Zxe2x80x94A1xe2x80x94; xe2x80x94Zxe2x80x94C(O)xe2x80x94A1xe2x80x94; xe2x80x94CH2xe2x80x94C(O)xe2x80x94A1xe2x80x94; xe2x80x94Zxe2x80x94C(O)xe2x80x94Zxe2x80x94A2xe2x80x94; xe2x80x94CH2xe2x80x94Zxe2x80x94C(O)xe2x80x94A2xe2x80x94;
xe2x80x94Zxe2x80x94CH2xe2x80x94C(O)xe2x80x94A2xe2x80x94; xe2x80x94Zxe2x80x94CH2xe2x80x94S(O)mxe2x80x94A2xe2x80x94; xe2x80x94C(O)xe2x80x94A3; xe2x80x94Zxe2x80x94A3xe2x80x94; or xe2x80x94A3xe2x80x94Zxe2x80x94;
X2 represents C2-3 alkylene, xe2x80x94C(O)xe2x80x94A4xe2x80x94 or xe2x80x94A4xe2x80x94C(O)xe2x80x94;
X3 represents CH or N;
X4 represents a single bond, O, S, C(O), N(R23), xe2x80x94CH(R23)xe2x80x94, xe2x80x94CH(R23)xe2x80x94CH(R24)xe2x80x94 or xe2x80x94C(R23)xe2x95x90C(R24)xe2x80x94;
A1 represents a single bond or C1-2 alkylene;
A2 represents a single bond or xe2x80x94CH2xe2x80x94;
A3 represents C1-3 alkylene;
A4 represents C(O) or C1-2 alkylene;
Z represents, at each occurrence, O, S(O)m or N(R25);
R2 and R4 independently represent one or more optional substituents selected from C1-4 alkyl, C1-4 alkoxy (which latter two groups are optionally substituted by one or more halo substituent), methlylenedioxy, halo, hydroxy, cyano, nitro, S(O)2NH2, C(O)OR26, SR26, S(O)R26a, S(O)2R26a or N(R27)R28;
R3 represents one or more optional substituents selected from OH, C1-4 alkoxy, C1-6 alkyl (optionally substituted by one or more halo group), or N(R29a)R29b;
R25, R29a and R29b independently represent H, C1-4 alkyl or C(O)R30;
R26 represents H or C1-4 alkyl;
R26a represents C1-4 alkyl;
R27 and R28 independently represent H, C1-4 alkyl or C(O)R30, or together represent C3-6 alkylene, thus forming a 4- to 7-membered ring, which ring is optionally substituted, on a carbon atom that is xcex1 to the nitrogen atom, with an xe2x95x90O group;
R21, R22, R23, R24 and R30 independently represent, at each occurrence, H or C1-4alkyl;
Y represents CH2, (CH2)2, CHxe2x95x90CH (which latter group is optionally substituted by C1-4 alkyl), (CH2)3, CH2CHxe2x95x90CH or CHxe2x95x90CHCH2 (which latter three groups are optionally substituted by C1-4 alkyl, methylene, xe2x95x90O or hydroxy);
Ry represents H or C1-4 alkyl;
n represents 0, 1, 2, 3 or 4; and
B represents a structural fragment of formula IIIa, IIIb or IIIc 
xe2x80x83wherein
X5, X6, X7 and X8 independently represent CH, N or Nxe2x80x94O;
X9 and X10 independently represent a single bond or CH2;
R31 represents an optional substituent selected from halo, C1-4 alkyl (which group is optionally substituted by one or more halo group), N(R32)R33, OR34 or SR35;
R32 and R33 independently represent H, C1-4 alkyl or C(O)R36; R34, R35 and R36 independently represent H or C1-4 alkyl; and
one of D1 and D2 represents H, and the other represents H, ORa, NHRa, C(xe2x95x90X11)X12Rb, or D1 and D2 together represent a structural fragment of formula IVa: 
Ra represents H or xe2x80x94A5[X14]n[C(O)]rRe;
Rb represents xe2x80x94A5[X14]n[C(O)]rRe;
A5 represents, at each occurrence, a single bond or C1-12 alkylene (which alkylene group is optionally interrupted by one or more O, S(O)m and/or N(Rf) group, and is optionally substituted by one or more of halo, OH, N(H)C(O)Rg, C(O)N(Rg)Rh, C3-7 cycloalkyl (which cycloalkyl group is optionally interrupted by one or more O, S(O)m and/or N(Rf) group and/or is optionally substituted by one or more substituents selected from C1-6 alkyl, C1-6 alkoxy, halo, xe2x95x90O or xe2x95x90S), Het and C6-10 aryl (which aryl and Het groups are themselves optionally substituted by one or more substituents selected from C1-6 alkyl (optionally substituted by one or more halo substituent), C1-6 alkoxy, halo, cyano, C(O)ORg, C(O)N(Rg)Rh and N(Rf)Rg));
Rc and Rd both represent H; or one of Rc and Rd represents H or C1-7 alkoxy and the other represents C1-7 alkyl (which alkyl group is optionally interrupted by one or more O atom); or Rc and Rd together represent C3-8 cycloalkyl, which cycloalkyl group is interrupted by one or more O, S(O)m and/or N(Rf) group;
Re represents, at each occurrence, H, C1-12 alkyl (which alkyl group is optionally interrupted by one or more O, S(O)m and/or N(Rf) group, and/or is optionally substituted by one or more substituents selected from halo, OH, N(H)C(O)Rg and C(O)N(Rg)Rh), A7xe2x80x94C3-7-cycloalkyl (which cycloalkyl group is optionally interrupted by one or more O, S(O)m and/or N(Rf) group and/or is substituted by one or more substituents selected from C1-6 alkyl, C1-6 alkoxy, halo, xe2x95x90O and xe2x95x90S), A7xe2x80x94C6-10 aryl or A7-Het (which aryl and Het groups are optionally substituted by one or more substituents selected from C1-6 alkyl (optionally substituted by one or more halo substituent), C1-6 alkoxy, halo, cyano, C(O)ORg, C(O N(Rg)Rh and N(Rf)Rg);
A7 represents a single bond or C1-7 alkylene (which alkylene group is optionally interrupted by one or more O, S(O)m and/or N(Rf) group, and/or are optionally substituted by one or more of halo, OH, N(H)CORg and CON(Rg)Rh);
Het represents, at each occurrence, a five- to ten-membered heteroaryl group, which may be aromatic in character, containing one or more nitrogen, oxygen or sulphur atoms in the ring system;
n and r independently represent 0 or 1;
X11, X12 and X14 independently represent O or S;
X13 represents O or NRf);
Rf represents, at each occurrence, H, C1-4 alkyl or C(O)Rg;
Rg and Rh independently represent, at each occurrence, H or C1-4 alkyl; and
m represents, at each occurrence, 0, 1 or 2;
or a pharmaceutically acceptable salt thereof;
provided that:
(a) A and E do not both represent O or S;
(b) E and D do not both represent O or S;
(c) when R1 represents OR1d and X1 represents xe2x80x94C(O)xe2x80x94Zxe2x80x94A1, xe2x80x94Zxe2x80x94CH2xe2x80x94S(O)mxe2x80x94A2xe2x80x94 or xe2x80x94Zxe2x80x94C(O)xe2x80x94Zxe2x80x94A2, then A1 or A2 (as appropriate) do not represent a single bond;
(d) when X4 represents xe2x80x94CH(R23)xe2x80x94, R1 does not represent OH;
(e) when A5 represents a single bond, then n and r both represent 0;
(f) when A5 represents C1-12 alkylene, then n represents 1;
(g) when A5 represents xe2x80x94CH2xe2x80x94, n is 1 and r is 0, then Re does not represent H; and
(h) the compound is not:
(S)- or (R)-1-hydroxy-7-methoxytetralin-1-yl-C(O)-Pro-Pab;
(R)- or (S)-1-hydroxy-7-methoxytetralin-1-yl-C(O)-Pro-Pab;
(S)- or (R)-1-hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab x HOAc;
(R)- or (S)-1-hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab;
1-hydroxy-5-methoxytetralin-1-yl-C(O)-Aze-Pab x HOAc;
1-hydroxy-5,7-dimethyltetralin-1-yl-C(O)-Aze-Pab x HOAc;
1-hydroxy-7-aminotetralin-1-yl-C(O)-Aze-Pab x HOAc;
1-hydroxytetralin-1-yl-C(O)-Aze-Pab x HOAc;
7-methoxytetralin-1-yl-C(O)-Aze-Pab x HOAc;
(R)- or (S)-7-methoxy-1-methyltetralin-1-yl-C(O)-Aze-Pab;,
4-hydroxy-6-methoxychroman-4-yl-C(O)-Aze-Pab x OAc;
(S)- or (R)-1-hydroxy-7-methoxyindan-1-yl-C(O)-Aze-Pab;
1-hydroxy-5-methoxytetralin-1-yl-C(O)-Aze-Pab(OH);
(S)- or (R)-1-hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab(OH);
4-hydroxy-6-methoxychroman-4-yl-C(O)-Aze-Pab(OH);
4-hydroxy-6-methoxychroman-4-yl-C(O)-Aze-Pab(OMe);
(S)- or (R)-1-hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab-(C(O)OCH2CHl3);
(S)- or (R)-1-hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab-(C(O)OCH2CH3);
7-methoxy-1-allyltetralin-1-yl-C(O)Aze-Pab x HOAc;
(S)- or (R)-1-hydroxy-7-chlorotetralin-1-yl-C(O)-Pro-Pab;
1-n-propyl-7-methoxytetralin-1-yl-C(O)-Aze-Pab x HOAc;
6-chloro-4-hydroxychroman-4-yl-C(O)Aze-Pab x HOAc;
4-hydroxychroman-4-yl-C(O)-Aze-Pab x HOAc;
6,8-dichloro-4-hydroxychroman-4-yl-C(O)-Aze-Pab x HOAc;
6-fluoro-4-hydroxychroman-4-yl-C(O)-Aze-Pab x HOAc;
4-hydroxy-6-methylchroman-4-yl-C(O)-Aze-Pab x HOAc;
8-chloro-4-hydroxy-6-methoxychroman-4-yl-C(O)-Aze-Pab x HOAc;
6-chloro-4-hydroxy-8-methylchroman-4-yl-C(O)-Aze-Pab x HOAc;
(S)- or (R)-1-hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab(O-C(O)-i-Pr);
(S)- or (R)-1-hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab(Q-C(O)-Et);
(S)- or (R)-1-hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab(Oxe2x80x94C(O)xe2x80x94Ch);
(S)- or (R)-1-hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab(O-allyl);
(S)- or (R)-1-hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab(O-Bzl);
(S)- or (R)-1-hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab-(COxe2x80x94O-methallyl);
1-hydroxy-7-aminotetralin-1-yl-C(O)-Aze-Pab(OH);
(S)- or (R)-1-hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-Pab(O-Val);
(S)- or (R)-1-hydroxy-7-methoxytetralin-1-yl-C(O)-Aze-(Me)Pab; or
9-hydroxyfluoren-9-yl-C(O)-Aze-Pab x HOAc,
which compounds are referred to hereinafter as xe2x80x9cthe compounds of the inventionxe2x80x9d.
The compounds of the invention may exhibit tautomerism. All tautomeric forms and mixtures thereof are included within the scope of the invention. Particular tautomeric forms of compounds of the invention that may be mentioned include those connected with the position of the double bond in the amidine functionality in the structural fragment B, and the position of D1 and D2, when one of these does not represent H, Further, it will be appreciated by those skilled in the art that, in the structural fragment of formula IIa, the optional. double bonds, may, in conjunction with certain identities of substituent D, render the ring bearing A, E and D aromatic in character.
The compounds of formula I may also contain one or more asymmetric carbon atoms and may therefore exhibit optical and/or diasteroisomerism. All diastereoisomers may be separated using conventional techniques, e.g. chromatography or fractional crystallisation. The various stereoisomers may be isolated by separation of a racemic or other mixture of the compounds using conventional, e.g. fractional crystallisation or HPLC, techniques. Alternatively the desired optical isomers may be made by reaction of the appropriate optically active starting materials under conditions which will not cause racemisation or epimerisation, or by derivatisation, for example with a homochiral acid followed by separation of the diastereomeric derivatives by conventional means (e.g. HPLC, chromatography over silica). All stereoisomers are included within the scope of the invention.
The term xe2x80x9carylxe2x80x9d includes phenyl, naphthyl and the like. Aryl groups may also be fused to cycloalkyl groups to form e.g. benzo-(C3-7)-cycloalkyl units (e.g. indanyl, indenyl, tetralinyl, and the like). The term xe2x80x9cHetxe2x80x9d includes groups such as pyridinyl, thiophenyl, furanyl, pyrrolidinyl, imidazolyl, indolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, oxatriazolyl, thiatriazolyl, pyridazinyl, morpholinyl, pyrimidinyl, pyrazinyl, quinolinyl, isoquinolinyl, piperidinyl, piperazinyl, chromanyl, thiochromanyl and the like.
Alkyl groups which R1, R1a, R1b, R1c, R1d, R2, R3, R4, R11, R12, R13, R14, R15, R16, R17, R18, R19, R21, R22, R23, R24, R25, R26, R26a, R27, R28, R29a, R29b, R30, R31, R32, R33, R34, R35, R36, Ry, Rf, Rg and Rh may represent, and with which Y, A5 and Re may be substituted; the alkyl part of alkylphenyl groups which R15 and R17 may represent; and alkoxy groups which R2, R3, R4, Rc and Rd may represent, and with which A5 and Re may be substituted, may, when there is a sufficient number of carbon atoms, be linear or branched, saturated or unsaturated, and/or cyclic, acyclic or part cyclic/acyclic. Alkyl groups which Rc, Rd and Re may represent, and alkylene groups which R27 and R28 (together), X1, X2, A1, A3, A4 and A7 may represent may, when there is a sufficient number of carbon atoms, be linear or branched, and/or saturated or unsaturated. Cycloalkyl groups which Rc and Rd may together represent, and which Re may include, may be branched and/or may be saturated or unsaturated.
Alkylene groups which A5 may represent may, when there is a sufficient number of carbon atoms, be linear or branched, be saturated or unsaturated, and/or be cyclic, acyclic or part cyclic/acyclic. The C3-7 cycloalkyl group with which A5 may be substituted, may be branched, saturated or unsaturated, and/or part cyclic/acyclic. This cycloalkyl group may also be attached to A5 via a carbon-carbon bond or may be attached directly to the alkylene chain (i.e. to give a xe2x80x9cspiroxe2x80x9d compound).
Halo groups, which R2, R4 and R31 may represent, and with which R1, R2, R3, R4, R31, A5, Re and A7 may be substituted, include fluoro, chloro, bromo and iodo.
In the structural fragments of formulae IIa, IIb and IIc, the dots indicate the carbon atom which is bonded to the xe2x80x94C(O)xe2x80x94 group and to R1 in a compound of formula I (for the avoidance of doubt, there is no further H atom bonded to the carbon atom so indicated).
The wavy lines on the bond in the fragments of formulae IIIa, IIIb, IIIc, IVa and Ar (below) signify the bond position of the fragment.
Abbreviations are listed at the end of this specification.
Preferred compounds of the invention include those in which, when:
R2 and R4 do not independently represent C1-4 alkoxy substituted by one or more halo substituent, SR26, S(O)R26a, S(O)2R26a or N(R27)R28, in which R27 and R28 independently represent C(O)R30, or together represent C3-6 alkylene, thus forming a 4- to 7-membered ring, which ring is optionally substituted, on a carbon atom that is xcex1 to the nitrogen atom, with a xe2x95x90O group, and R26, R26a and R30 are as hereinbefore defined;
R3 does not represent one or more optional substituents selected from C1-6 alkyl (optionally substituted by one or more halo group) or N(R29a)R29b, in which R29a and R29b are as hereinbefore defined;
R25 does not represent C(O)R30, in which R30 is as hereinbefore defined;
Y does not represent CHxe2x95x90CH substituted by C1-4 alkyl; and/or
R31 does not represent C1-4 alkyl (substituted by one or more halo group), N(R32)R33, OR34 or SR35, in which R32, R33, R34 and R35 are as hereinbefore defined, (i.e. when the values of R2, R4, R3, R25, Y and R31 are other than those listed immediately above)
then
(i) D1 and D2 do not both represent H;
(ii) when D1 or D2 represents ORa, then Ra does not represent H, phenyl, benzyl or C1-7 alkyl (which latter group is optionally interrupted by O or is optionally substituted by halo);
(iii) when D1 or D2 represents C(X11)X12Rb and X11 and X12 both represent O, then Rb does not represent 2-naphthyl, phenyl, C1-3 alkylphenyl (which latter three groups are optionally substituted by C1-6 alkyl, C1-6 alkoxy or halo); C1-12 alkyl (which latter group is optionally substituted by C1-6 alkoxy, C1-6 acyloxy or halo); xe2x80x94[C(Rq)(Rr)]pOC(O)Rs, in which p is 1, 2 or 3, Rq and Rr independently represent H or C1-6 alkyl (provided that the total number of carbon atoms in [C(Rq)(Rr)]p does not exceed 12), and Rs represents C1-6 alkyl (optionally substituted by C1-6 alkoxy), C1-12 alkyl (optionally substituted by halo), C3-7 cycloalkyl, phenyl, naphthyl or C1-3 alkylphenyl (which latter four groups are optionally substituted by C1-6 alkyl or halo); or xe2x80x94CH2-Ar, in which Ar represents the structural fragment: 
Compounds of the invention which may be mentioned include those in which:
R2 and R4 independently represent C1-4 alkoxy substituted by one or more halo substituents, SR26, S(O)R26a, S(O)2R26a or N(R27)R28, in which R27 and R28 independently represent C(O)R30, or together represent C3-6 alkylene, thus forming a 4- to 7-membered ring, which ring is optionally substituted, on a carbon atom that is xcex1 to the nitrogen atom, with a xe2x95x90O group, and R26, R26a and R30 are as hereinbefore defined;
R3 represents one or more optional substituents selected from C1-6 alkyl (optionally substituted by one or more halo group) or N(29a)R29b, in which R29a and R29b are as hereinbefore defined;
R25 represents C(O)R30, in which R30 is as hereinbefore defined;
Y represents CHxe2x95x90CH substituted by C1-4 alkyl;
R31 represents C1-4 alkyl (substituted by one or more halo group), N(R32)R33, OR34 or SR35, wherein R32, R33, R34 and R35 are as hereinbefore defined.
Further compounds of the invention which may be mentioned include those in which:
(i) when one of D1 or D2 represents ORa, then Ra does not represent H, phenyl, benzyl or C1-7 alkyl (which latter group is optionally interrupted by O or is optionally substituted by halo);
(ii) when one of D1 or D2 represents C(X11)X12Rb and X11 and X12 both represent O, then Rb does not represent 2-naphthyl, phenyl, C1-3 alkylphenyl (which latter three groups are optionally substituted by C1-6 alkyl, C1-6 alkoxy or halo); C1-12 alkyl (which latter group is optionally substituted by C1-6 alkoxy, C1-6 acyloxy or halo); xe2x80x94[C(Rq)(Rr)]pOC(O)Rs, in which p is 1, 2 or 3, Rq and Rr independently represent H or C1-6 alkyl (provided that the total number of carbon atoms in [C(Rq)(Rr)]p does not exceed 12), and Rs represents C1-6 alkyl (optionally substituted by C1-6 alkoxy), C1-12 alkyl (optionally substituted by halo), C3-7 cycloalkyl, phenyl, naphthyl or C1-3 alkylphenyl (which latter four groups are optionally substituted by C1-6 alkyl or halo); or xe2x80x94CH2-Ar, in which Ar represents the structural fragment: 
When n represents 2and B represents a structural fragment of formula IIIb, preferred compounds of the invention include those wherein X9 and X10 do not both represent CH2.
Preferred compounds of formula I include those wherein:
R1 represents OH or C1-4 alkyl (which latter group is optionally substituted by cyano or OH);
Rx represents a structural fragment of formula IIb or, especially, IIa;
when Rx represents a structural fragment of formula IIa, the dotted lines represent bonds, A and E both represent CH and D represents xe2x80x94CH xe2x95x90CHxe2x80x94;
when Rx represents a structural fragment of formula IIa, X1 represents optionally unsaturated C2- or C3-alkylene, or xe2x80x94Zxe2x80x94A3 (in which Z represents O, S(O)m or N(R25) (in which R25 is as hereinbefore defined or represents C1-4 alkyl or C(O)R30 and m and R30 are as hereinbefore defined) and A3 represents C1- or C2-alkylene (which latter group is optionally unsaturated));
Y represents (CH2)3, preferably (CH2)2 and more preferably CH2;
B represents a structural fragment of formula IIIa in which X5, X6, X7 and X8 all represent CH.
Particularly preferred compounds of the invention include those wherein, when Rx represents a structural fragment of formula IIa, X1 represents C3-alkylene or xe2x80x94Z(CH2)2xe2x80x94, in which Z represents S(O)m, N(R25) (in which R25 is as hereinbefore defined) or, especially, O.
When Rx represents a structural fragment of formula IIa, and R2 represents at least one substituent, a preferred point of substitution is at the carbon atom which is at position E. It is preferred that at least one (and preferably two) substituents R2 are present in a structural fragment of formula IIa.
When Rx represents a structural fragment of formula IIa, the dotted lines represent bonds, A and E both represent CH and D represents xe2x80x94CHxe2x95x90CHxe2x80x94 (i.e. the ring bearing R2 is a benzo group), and R2 represents at least one substituent, the ring is preferably substituted either at the carbon atom in the xe2x80x94CHxe2x95x90CHxe2x80x94 group (position D) which is adjacent to the ring junction, or at the carbon atom which is at position E, or preferably at both of these sites. For example, when the fragment IIa represents a tetralin-1-yl group (i.e. the dotted lines represent bonds, A and E both represent CH, D represents xe2x80x94CHxe2x95x90CHxe2x80x94 and X1 represents saturated C3-alkylene), preferred substitution positions are the 5- and 7-positions, or, preferably, di-substitution at both of these positions. Correspondingly, when the fragment IIa represents a chroman-4-yl, a thiochroman-4-yl, or a quinolin-4-yl, group (i.e. the dotted lines represent bonds, A and E both represent CH, D represents xe2x80x94CHxe2x95x90CHxe2x80x94, and X1 represents xe2x80x94Z(CH2)2xe2x80x94, in which Z represents O, S(O)m or N(R25)), preferred substitution positions are the 8- and 6-positions, or, preferably, di-substitution at both of these positions.
Preferred optional substituents R2 include halo, C1-4 alkyl, C1-4 alkoxy (which latter two groups are optionally substituted by one or more halo groups) or N(R27)R28.
When R1 represents OH, Rx represents an unsubstituted (by R2 and R4) structural fragment of formula IIc, in which X4 represents a single bond, CH2 or O, Y represents CH2 or (CH2)2, Ry represents H and n represents 1, preferred compounds of the invention include those in which B does not represent a structural fragment of formula IIIb in which X9 and X10 are both CH2 and D1 and D2 are both H.
When D1 and D2 together represent a structural fragment of formula IVa, in which X13 is O, preferred compounds of the invention include those in which one of Rc and Rd represents H or C1-7 alkoxy and the other represents C1-7 alkyl (e.g. C1-4 alkyl, including linear, saturated, unsubstituted, and uninterrupted, C1-4 alkyl).
When D1 or D2 represents ORa and Ra represents xe2x80x94A5[X14]n[C(O)]rRe, and:
(i) A5 is a single bond (and thus n and r both represent 0), preferred compounds of the invention include those in which Re is:
(1) optionally substituted A7-aryl, in which A7 is preferably a single bond or C1-3 alkylene (e.g. C1-2-alkylene) and aryl is preferably C6-10-aryl, (e.g. phenyl), which A7-aryl group is optionally substituted by one or more halo, C1-6 alkoxy (e.g. C1-4 alkoxy, such as methoxy), C1-6 alkyl (e.g. C1-4 alkyl) or a haloalkyl (e.g. CF3) substituent);
(2) H or linear, branched, optionally unsaturated, and/or cyclic, C1-12 alkyl (e.g. C3-7 alkyl), which cyclic alkyl group is optionally interrupted by an O atom and, optionally, a further O atom or S(O)m group;
(ii) A5 is linear or branched C1-2 alkylene, X14 is O and r is 0, preferred compounds of the invention include those in which Re is C1-3 alkyl or A7-aryl, in which A7 is a single bond and the aryl group is preferably optionally substituted phenyl.
When D1 or D2 represents ORa, preferred compounds of the invention include those in which Ra is H or C1-4, alkyl.
When D1 or D2 represents xe2x80x94C(xe2x95x90X11)X12Rb, in which X11 represents O and X12 represents O or S, and, in which Rb group, A5 represents a single bond (and thus n and r both represent 0), preferred compounds of the invention include those in which Re represents optionally unsaturated C1-6 (e.g. C1-4) alkyl, A7xe2x80x94C6-10-aryl (in which A7 represents a single bond or C1-2 alkylene and the C6-10 aryl group is preferably phenyl, which A7xe2x80x94C6-10-aryl group is optionally substituted by one or more halo, C1-4 alkyl and/or C1-4 alkoxy groups), or A7xe2x80x94C3-7-cycloalkyl (especially A7xe2x80x94C4-5 cycloalkyl), in which A7 represents a single bond or linear or branched C1-7 alkylene, and which cycloalkyl group is optionally substituted by C1-3 alkyl.
Compounds of formula I in which the fragment 
is in the S-configuration are preferred. The wavy lines on the bonds in the above fragment signify the bond position of the fragment.
Preferred compounds of formula I include the compounds of the Examples described hereinafter.
Preparation
According to the invention there is also provided a process for the preparation of compounds of formula I which comprises:
(i) the coupling of a compound of formula IV, 
xe2x80x83wherein R1 and Rx are as hereinbefore defined with a compound of formula V, 
xe2x80x83wherein Ry, Y, n and B are as hereinbefore defined, for example in the presence of a coupling agent (e.g. oxalyl chloride in DMF, PyBOP, EDC, DCC, HBTU, HATU or TBTU), an appropriate base (e.g. pyridine, 2,4,6-trimethylpyridine, 2,4,6-collidine, DMAP, TEA or DIPEA) and a suitable organic solvent (e.g. dichloromethane, acetonitrile or DMF);
(ii) the coupling compound of formula VI, 
xe2x80x83wherein R1, Rx and Y are as hereinbefore defined with a compound of formula VII,
H(Ry)Nxe2x80x94(CH2)nxe2x80x94Bxe2x80x83xe2x80x83(VII)
xe2x80x83wherein Ry, n and B are as hereinbefore defined, for example in the presence of a coupling agent (e.g. oxalyl chloride in DMF, PYBOP, EDC, DCC, HBTU, HATU or TBTU), an appropriate base (e.g. pyridine, 2,4,6-trimethylpyridine, 2,4,6-collidine, DMAP, TEA or DIPEA) and a suitable organic solvent (e.g. dichloromethane, acetonitrile or DMF);
(iii) for compounds of formula I in which D1 or D2 represents ORa or NHRa, reaction of a compound of formula VIII, 
xe2x80x83wherein B1 represents a structural fragment of formula IIId, IIIe or IIIf 
xe2x80x83and R1, Rx, Y, Ry, n, R31, X5, X6, X7, X8, X9 and X10 are as hereinbefore defined with a compound of formula IX,
H2NXaRaxe2x80x83xe2x80x83IX
xe2x80x83wherein Xa represents O or NH and Ra is as hereinbefore defined, for example at between 40 and 70xc2x0 C. (e.g. 60xc2x0), in the presence (optionally) of a suitable base (e.g. TEA), and an appropriate organic solvent (e.g. THF, CH3CN, DMF or DMSO), and, optionally, wherein the compound of formula VIII is first treated with gaseous HCl, in the presence of a lower alkyl alcohol (e.g. ethanol) at, for example, 0xc2x0 C.;
(iv) for compounds of formula I in which D1 or D2 represents ORa or NHRa, reaction of a compound of formula I in which D1 or D2 (as appropriate) represents C(O)ORb1, in which Rb1 represents a protecting group (such as a 2-trimethylsilylethyl, a suitable alkyl (e.g. C1-6 alkyl), or alkylphenyl (e.g. benzyl), group) with a compound of formula IX as hereinbefore defined, for example under similar reaction conditions to those described hereinbefore for preparation of compounds of formula I (step (iii)) (the skilled person will appreciate that in such a reaction the diprotected (i.e. C(O)ORb1 and ORa/NHRa protected) derivative may, in some cases, be isolated if desired, and the C(O)ORb1 group then removed using conventional techniques);
(v) for compounds of formula I in which D1 or D2 represents ORa or NHRa, Ra represents xe2x80x94A5[X14]n[C(O)]rRe, in which A5 does not represent a single bond, and n represent 1, reaction of a compound of formula I in which D1 or D2 (as appropriate) represents OH or NH2, with a compound of formula X,
L1A5a[X14][C(O)]rRexe2x80x83xe2x80x83X
xe2x80x83wherein L1 represents a suitable leaving group, such as lower alkoxy or halo, A5a represents A5, as hereinbefore defined except that it does not represent a single bond, and X14, r and Re are as hereinbefore define, for example under conditions that are well known to those skilled in the art (see e.g. U.S. Pat. No. 3,822,283);
(vi) for compounds of formula I in which D1 or D2 represents ORa or NHRa, Ra represents xe2x80x94A5[X14]n[C(O)]rRe, in which A5 represents C2-12 alkylene, which alkylene group is branched at the carbon atom that is xcex1 to the O or N atom of ORa or NHRa (as appropriate), and which group is optionally branched at the carbon atom that is xcex2 to that atom, n represents 1, r represents 0 and Re is as hereinbefore defined, reaction of a compound of formula I in which D1 or D2 (as appropriate) represents OH or NH2, with a compound of formula XI, 
xe2x80x83or a geometrical isomer thereof, or a mixture of such geometrical isomers, in which Rb1 and Rb3 each represent H or an alkyl group, provided that the total number of carbon atoms provided by Rb1 and Rb3 does not exceed 10, and wherein X14 and Re are as hereinbefore define, for example under conditions that are well known to those skilled in the art;
(vii) for compounds of formula I in which D1 or D2 represents ORa or NHRa, Ra represents xe2x80x94A5[X14]n[C(O)]rRe, in which A5 represents a single bond (and thus n and r both represent 0), and Re represents A7xe2x80x94C3-6-cycloallyl, in which A7 represents a single bond, and the cycloalkyl group is interrupted by at least one O or S atom, which atom is between the carbon atom at the point of attachment to the O or NH group of ORa or NHRa, and a carbon atom that is xcex1 to that point of attachment, and which cycloalkyl group is optionally interrupted by one or more O or S(O)m group and/or optionally substituted by one or more xe2x95x90O group, reaction of a compound of formula I, in which D1 or D2 (as appropriate) represents OH or NH2, with a compound of formula XII, 
xe2x80x83wherein X15 represents O or S and X16 represents C1-4 alkylene (which alkylene group is optionally interrupted by one or more O or S(O)m group and/or optionally substituted by one or more xe2x95x90O group), for example under conditions that are well known to those skilled in the art;
(viii) for compounds of formula I in which D1 or D2 represents C(X11)X12Rb, reaction of a compound of formula I in which D1 and D2 both represent H with a compound of formula XIII,
L2C(X11)X12Rbxe2x80x83xe2x80x83XIII
xe2x80x83wherein L2 represents a suitable leaving group, such as halo or p-nitrophenoxy, and X11, X12 and Rb are as hereinbefore define, for example 0xc2x0 C. in the presence of a suitable base (e.g. NaOH) and an appropriate organic solvent (e.g. THF) or water;
(ix) for compounds of formula I in which D1 and D2 together represent a structural fragment of formula IVa, reaction of a corresponding compound of formula I in which D1 or D2 represents OH or NHRf (in which Rf is as hereinbefore defined), with a compound of formula XV,
(Rc)(Rd)C(Rc1)(Rc2)xe2x80x83xe2x80x83XV
xe2x80x83wherein Rc1 and Rc2 both represent xe2x80x94ORc3, in which Rc3 represents C1-3 alkyl, or together represent xe2x95x90O, and Rc and Rd are as hereinbefore defined, for example by using the compound of formula XV as solvent and HCl as a catalyst, at between room temperature and reflux (see e.g. J. Org. Chem. USSR, 21, 177 (1985));
(x) for compounds of formula I in which one or more of X5, X6, X7 and X8 represent Nxe2x80x94O, oxidation of a corresponding compound of formula I in which X5, X6, X7 and/or X8 (as appropriate) represent(s) N under conditions that are well known to those skilled in the art (for example in the presence of a suitable oxidising agent (e.g. mCPBA), at an appropriate temperature (e.g. 0xc2x0 C.), and in the presence of a suitable organic solvent (e.g. DCM));
(xi) for compounds of formula I in which any one of Z, X1, R2, R4, A5, A7, Rc, Rd and/or Re comprises or includes a S(O) or a S(O)2 group, oxidation of a corresponding compound of formula I (or a compound corresponding to a compound of formula I) wherein Z, X1, R2, R4, A5, A7, Rc, Rd and/or Re (as appropriate) comprise(s) or include(s) a S group, in the presence of an appropriate amount of a suitable oxidising agent (e.g. mCPBA) and an appropriate organic solvent; or
(xii) for compounds of formula I in which D1 and D2 both represent H, removal of a ORa, NHRa or C(xe2x95x90X11)X12Rb group (in which Ra, Rb, X11 and X12 are as hereinbefore defined), or removal of a structural fragment of formula IVa as hereinbefore defined, from a corresponding compound of formula I (i.e. deprotection) under conditions known to those skilled in the art.
Compounds of formula IV are commercially available, are well known in the literature, or are available using known and/or standard techniques.
For example, compounds of formula IV in which R1 represents OH may be prepared by reaction of a compound of formula XVI,
Rx=Oxe2x80x83xe2x80x83XVI
wherein Rx is as hereinbefore defined, with:
(a) KCN, for example at 20xc2x0 C. in the presence of sodium bisulphite in water, followed by hydrolysis in the presence of aqueous acid (e.g. HCl), for example at 20xc2x0 C. in the presence of a suitable solvent (e.g. alcohol and/or water);
(b) CHCl3, in the presence of aqueous base (e.g. NaOH);
(c) TMSCN, for example at 20xc2x0 C. in the presence of a suitable organic solvent (e.g. CH2Cl2), followed by hydrolysis in the presence of acid (e.g. HCl or H2SO4), for example at 20xc2x0 C. (e.g. according, or analogously, to the method described by Bigge et al in J. Med. Chem. (1993) 36, 1977), followed by alkaline hydrolysis to give the free acid.
Compounds of formula IV in which R1 represents OH may alternatively be prepared by way of a Sharpless stereoselective dihydroxylation of a compound of formula XVIA,
Rx=CH2xe2x80x83xe2x80x83XVIA
wherein Rx is as hereinbefore define, under conditions known to those skilled in the art (e.g. at low temperature (e.g. 0xc2x0 C.), using, for example, the commercial reagent AD-mix-xcex2(trademark) in the presence of suitable solvent (e.g. t-butanol), followed by oxidation of the resultant intermediate (e.g. at elevated temperature (e.g. 75xc2x0 C.) in the presence of a stream of air and Pt/C (5%) in acetone/water).
Compounds of formula IV in which R1 represents H may be prepared from corresponding compounds of formula IV in which R1 represents OH (or a lower alkyl ester of the acid), for example by elimination of water, followed by hydrogenation of the resultant alkene using techniques which are well known to those skilled in the art, followed by, if necessary, hydrolysis to give the free acid.
Compounds of formula IV in which R1 represents C1-4 alkyl may be prepared from corresponding compounds of formula IV in which R1 represents H (or a lower alkyl ester of the acid), for example by reaction with an appropriate alkyl halide using techniques which are well known to those skilled in the art, followed by, if necessary, hydrolysis to give the free acid.
Compounds of formula IV in which R1 represents OR1d and R1d represents C(O)R11, SiR12R13R14 or C1-6 alkyl may be prepared by acylation, silylation or alkylation (as appropriate) of a corresponding compound of formula IV in which R1 represents OH (or a lower alkyl ester of the acid) under conditions which are well known to those skilled in the art, followed by, if necessary, hydrolysis to give the free acid.
Compounds of formula V may be prepared by reaction of a compound of formula XVII 
wherein Y is as hereinbefore defined with a compound of formula VII as hereinbefore defined, for example under conditions such as those described hereinbefore for synthesis of compounds of formula I.
Compounds of formulae V and VII in which Ry represents C1-4 alkyl may be prepared by reaction of a corresponding compound of formula V or formula VII, as appropriate, in which Ry represents H with a compound of formula XVIII,
RyHalxe2x80x83xe2x80x83XVIII
wherein Hal represents halo (e.g. Cl, Br or I) and Ry is as hereinbefore defined, for example under conditions which are well known to those skilled in the art.
Compounds of formula VI are readily available using known techniques. For example, compounds of formula VI may be prepared by reaction of a compound of formula IV as hereinbefore defined with a compound of formula XVII as hereinbefore defined, for example under conditions such as those described hereinbefore for synthesis of compounds of formula I.
Compounds of formula VIII may be prepared in accordance with peptide coupling techniques, for example in analogous fashion to the methods described hereinbefore for compounds of formula I.
Compounds of formula XVI are commercially available, are well known in the literature, or may be prepared in accordance with known techniques. For example compounds of formula XVI may be prepared as follows:
(a) Compounds of formula XVI in which Rx represents a structural fragment of formula IIa, in which the dotted lines represent bonds, A and E both represent CH and D represents xe2x80x94CHxe2x95x90CHxe2x80x94; X1 represents C2-4 alkylene, xe2x80x94Zxe2x80x94A3xe2x80x94 or xe2x80x94C(O)xe2x80x94A3xe2x80x94, in which Z and A3 are as hereinbefore defined; and R3 is absent, may be prepared by cyclisation of a compound of formula XIX, 
xe2x80x83wherein X1a represents C2-4 alkylene, xe2x80x94Zxe2x80x94A3xe2x80x94 or xe2x80x94C(O)xe2x80x94A3xe2x80x94, and Z, A3 and R2 are as hereinbefore defined, using an appropriate acylating agent, for example at 100xc2x0 C. in the presence of polyphosphoric acid or using PCl5 followed by AlCl3, or at low temperature (e.g. 5xc2x0 C.) in the presence of boron trifluoride dimethyl etherate and/or trifluoroacetic anhydride and an appropriate solvent (e.g. CH2Cl2). Compounds of formula XIX in which X1a represents C3-alkylene or xe2x80x94C(O)xe2x80x94A3xe2x80x94, in which A3 represents C2-alkylene, may be prepared in accordance with known techniques, for example by reaction of succinic anhydride with the corresponding phenyl lithium and, for compounds of formula XIX in which X1a represents C3-alkylene, selective reduction of the resultant ketone, under conditions which are well known to those skilled in the art. Compounds of formula XIX in which X1a represents xe2x80x94Zxe2x80x94A3xe2x80x94 and A3 represents C2-3 alkylene may be prepared as described hereinafter.
(b) Compounds of formula XVI in which Rx represents a structural fragment of formula IIa, in which the dotted lines represent bonds, A and E both represent CH and D represents xe2x80x94CHxe2x95x90CHxe2x80x94; X1 represents C2-4 alkylene or xe2x80x94C(O)xe2x80x94A3xe2x80x94, in which A3 is as hereinbefore defined; and R3 is absent, may alternatively be prepared by cyclisation of a compound of formula XX, 
xe2x80x83wherein R represents C1-6 alkyl and X1a and R2 are as hereinbefore defined, for example at 20xc2x0 C. in the presence of a suitable base (e.g. an alkali metal alkoxide) and an appropriate organic solvent (e.g. lower alkyl alcohol) followed by hydrolysis and decarboxylation. Compounds of formula XX may be prepared in accordance with known techniques. For example, compounds of formula XX in which X1a represents C3-alkylene or xe2x80x94C(O)xe2x80x94A3xe2x80x94 in which A3 represents C2-alkylene may be prepared by reaction of succinic anhydride with a compound of formula XXI, 
xe2x80x83wherein Rxe2x80x2 represents C1-6 alkyl and R and R2 are as hereinbefore defined and, for compounds of formula XX in which X1a represents C3-alkylene, selective reduction of the resultant ketone, followed by functional group transformations of the amide and the acid to ester groups, under conditions which are well known to those skilled in the art.
(c) Compounds of formula XVI in which Rx represents a structural fragment of formula IIa, in which the dotted lines represent bonds, A and E both represent CH and D represents xe2x80x94CHxe2x95x90CHxe2x80x94; X, represents xe2x80x94Zxe2x80x94A3xe2x80x94 in which A3 represents C2 alkylene and Z represents O or S; and R3 is absent, may be prepared by cyclisation of a compound of formula XXII, 
xe2x80x83wherein Za represents O or S and Hal and R2 are as hereinbefore defined, for example at 20xc2x0 C. in the presence of aqueous-ethanolic NaOH, For to corresponding compounds of formula XVI in which X1 represents xe2x80x94Zxe2x80x94A3xe2x80x94 and Z represents S(O)m in which m is 1 or 2, this above-mentioned cyclisation should be followed by carrying out an oxidation reaction on the cyclised product comprising an S atom, for example using m-chloroperbenzoic acid.
(d) Compounds of formula XVI in which Rx represents a structural fragment of formula IIa, in which the dotted lines represent bonds, A and E both represent CH and D represents xe2x80x94CHxe2x95x90CHxe2x80x94; X1 represents xe2x80x94Zxe2x80x94A3xe2x80x94 (in which A3 represents C2-alkylene) or xe2x80x94Zxe2x80x94C(O)xe2x80x94A1 (in which A1 represents C1-alkylene); and R3 is absent, may be prepared by reaction of a compound of formula XXIII, 
xe2x80x83wherein R2 and Z are as hereinbefore defined, with either:
(1) for compounds of formula XVI in which X1 represents xe2x80x94Zxe2x80x94A3xe2x80x94 in which A3 represents C2-alkylene, a compound of formula XXIV,
H2Cxe2x95x90CHxe2x80x94CO2Rxe2x80x83xe2x80x83XXIV
xe2x80x83wherein R is as hereinbefore defined, for example at 20xc2x0 C. in the presence of a suitable base (e.g. triethylamine or sodium ethoxide) and an appropriate organic solvent (e.g. ethanol or DMF); or
(2) a compound of formula XXV,
L1xe2x80x94Gxe2x80x94CH2xe2x80x94CO2Rxe2x80x83xe2x80x83XXV
xe2x80x83wherein L1 represents a suitable leaving group (such as Cl, Br, I, mesylate or tosylate), G represents CH2 or C(O) and R is as hereinbefore defined, for example at 20xc2x0 C. in the presence of a suitable base (e.g. triethylamine) and an appropriate organic solvent (e.g. THF); followed by cyclisation under appropriate conditions (e.g. those described hereinbefore).
(e) Compounds of formula XVI in which Rx represents a structural fragment of formula IIa, in which the ring bearing A, E and D is a carbocyclic aromatic, or heterocyclic aromatic, ring as defined hereinbefore in respect of compounds of formula I; X1 represents xe2x80x94CH2xe2x80x94Zxe2x80x94C1-2 alkylene-, in which Z is as hereinbefore defined; and R3 is absent, may be prepared by reaction of a compound of formula XXVI, 
xe2x80x83wherein the ring bearing Aa, Ea and Da is a carbocyclic aromatic, or heterocyclic aromatic, ring as define hereinbefore in respect of compounds of formula I, and Z and R2 are as hereinbefore defined, with a compound of formula XXVII,
L1-Alk-CO2Hxe2x80x83xe2x80x83XXVII
xe2x80x83wherein Alk represents C1-2 alkylene and L1 is as hereinbefore defined, for example at 20xc2x0 C. in the presence of a suitable base (e.g. sodium methoxide) and an appropriate organic solvent (e.g. THF).
(f) Compounds of formula XVI in which Rx represents a structural fragment of formulae IIb, IIc or IIa, in which latter case the ring bearing A, E and D is a carbocyclic aromatic, or heterocyclic aromatic, ring as defined hereinbefore in respect of compounds of formula I; and, in the cases when Rx represents a structural fragment of formulae IIa or IIb, R3 is absent, may be prepared by cyclisation of a compound of formula XXIX,
Rxaxe2x80x94CO2Hxe2x80x83xe2x80x83XXIX
xe2x80x83wherein Rxa represents a structural fragment of formula XXIXa, XXIXb or XXIXc 
xe2x80x83wherein, in XXIXa, the ring bearing Aa, Ea and Da is a carbocyclic aromatic, or heterocyclic aromatic, ring as defined hereinbefore in respect of compounds of formula I, and R2, R4, X1, X2, X3 and X4 are as hereinbefore defined, in the presence of polyphosphoric acid, for example at 100xc2x0 C. The dots adjacent to the carbon atoms in fragments of formula XXIXa, XXIXb and XXIXc signify the point of attachment of the fragments to the CO2H group of the compound of formula XXIX. Compounds of formula XXIX may be prepared by hydrolysis of a corresponding compound of formula XXX,
Rxaxe2x80x94CO2Rxe2x80x83xe2x80x83XXX
xe2x80x83wherein Rxa and R are as hereinbefore defined (and in which the CO2H in the fragments of formulae XXIXa, XXIXb and XXIXc in Rxa may also be replaced by CO2R), for example under reaction conditions which are well known to those skilled in the art.
(g) Compounds of formula XVI in which Rx represents a structural fragment of formula IIa in which the ring bearing A, E and D is a carbocyclic aromatic, or heterocyclic aromatic, ring as defined hereinbefore in respect of compounds of formula I; X1 represents xe2x80x94OCH2xe2x80x94; and R3 is absent, may be prepared by reaction of a compound of formula XXXI, 
xe2x80x83wherein the ring bearing Aa, Ea and Da is a carbocyclic aromatic, or heterocyclic aromatic, ring as defined hereinbefore in respect of compounds of formula I, and R2, Hal and R are as hereinbefore defined, with diazomethane, for example at 20xc2x0 C. in the presence of a suitable organic solvent (e.g. diethyl ether).
(h) Compounds of formula XVI in which Rx represents a structural fragment of formula IIa, in which the dotted lines represent bonds, A and E both represent CH and D represents xe2x80x94CHxe2x95x90CHxe2x80x94; X1 represents xe2x80x94C(O)xe2x80x94Oxe2x80x94CH2xe2x80x94; and R3 is absent, may be prepared by cyclisation of a compound of formula XXXII, 
xe2x80x83wherein R2 and R are as hereinbefore defined, for example at xe2x88x9220xc2x0 C. in the presence of sulphuric acid and an appropriate organic solvent (e.g. methanol). Compounds of formula XXXII may be prepared by reacting a corresponding acid halide with diazomethane, for example at 20xc2x0 C. in the presence of a suitable organic solvent (e.g. diethyl ether).
(i) Compounds of formula XVI in which Rx represents a structural fragment of formula IIa, in which X1 includes N(R25), or IIc, in which X4 represent N(R23), (as appropriate), and R23 and R25 (as appropriate) represent C1-4 alkyl, may be prepared by reaction of a corresponding compound of formula XVI in which X1 includes, or X4 represents, (as appropriate) NH with a compound of formula XXXIII
Raxe2x80x94Halxe2x80x83xe2x80x83XXXIII
xe2x80x83wherein Ra represents C1-4 alkyl and Hal is as hereinbefore define, for example under conditions which are well known to those skilled in the art.
(j) Compounds of formula XVI in which Rx represents a structural fragment of formula IIa, in which the dotted lines represent bonds, A and E both represent CH and D represents xe2x80x94CHxe2x95x90CHxe2x80x94; X1 represents xe2x80x94C(O)xe2x80x94N(H)xe2x80x94CH2xe2x80x94; and R3 is absent, may be prepared by catalytic hydrogenation of an hydroxamic acid of formula XXXIV, 
xe2x80x83wherein R2 is as hereinbefore defined, using an appropriate catalyst system (e.g. Pd/C) in the presence of a suitable organic solvent (e.g. methanol). Compounds of formula XXXIV may be prepared by cyclisation of a corresponding compound of formula XXXV, 
xe2x80x83wherein R2 is as hereinbefore defined, for example at 20xc2x0 C. in the presence of fuming HCl and tin dichloride.
(k) Selective oxidation of a compound of formula XXXVI,
Hxe2x80x94Rxxe2x80x94Hxe2x80x83xe2x80x83XXXVI
xe2x80x83wherein Rx is as hereinbefore defined, for example in the presence of a suitable oxidising agent (e.g. CrO3 or KMnO4) and an appropriate solvent (e.g. water).
(l) Selective oxidation of a compound of formula XXXVII,
Hxe2x80x94Rxxe2x80x94OHxe2x80x83xe2x80x83XXXVII
xe2x80x83wherein Rx is as hereinbefore defined, for example in the presence of a suitable oxidising agent (e.g. MnO2) in an appropriate organic solvent (e.g. CH2Cl2).
(m) Hydrolysis of an oxime formula XXXVIII,
Rxxe2x95x90Nxe2x80x94OHxe2x80x83xe2x80x83XXXVIII
xe2x80x83wherein Rx is as hereinbefore define, for example by heating in the presence of acid (e.g. HCl) and an appropriate organic solvent. Compounds of formula XXXVIII may be prepared by reaction of a corresponding compound of formula XXXVI, as hereinbefore defined, with propyl nitrite, for example in the presence of HCl in ethanol.
(n) Compounds of formula XVI in which Rx represents a structural fragment of formula IIa and X1 represents xe2x80x94CH2xe2x80x94CHxe2x95x90CHxe2x80x94, may be prepared by elimination of a compound of formula XXXIX, 
xe2x80x83wherein L3 represents a suitable leaving group (e.g. Br or SePh) and the dotted lines, A, E, D, R2 and R3 are as hereinbefore define, under appropriate reaction conditions, for example in the presence of aqueous ethanolic NaOH or hydrogen peroxide, and an appropriate organic solvent (e.g. THF).
(o) Compounds of formula XVI in which Rx represents a structural fragment of formula IIb, X2 represents xe2x80x94C(O)xe2x80x94A4xe2x80x94 and A4 is as hereinbefore define, may be prepared by cyclisation of a compound of formula XL, 
xe2x80x83wherein Rb represents OH, C1-6 alkoxy or Hal and R2, R3, A4, X3 and Hal are as hereinbefore defined, for example in the presence of polyphosphoric acid, as described hereinbefore or, in the case where Rb represents Hal, in the presence of AlCl3 in nitromethane at, for example, 20xc2x0 C.
(p) Compounds of formula XVI in which Rx represents a structural fragment of formula IIb and X2 represents xe2x80x94A4xe2x80x94C(O)xe2x80x94 and A4 represents C1-2 alkylene may be prepared by cyclisation of a compound of formula XLI, 
xe2x80x83wherein A4a represents C1-2 alkylene and Hal, R2, R3 and X3 are as hereinbefore define.
Compounds of formulae VII, IX, X, XI, XII, XIII, XV, XVIA, XVII, XVIII, XXI, XXII, XXIII, XXIV, XXV, XXVI, XXVII, XXX, XXXI, XXXIII, XXXV, XXXVI, XXXVII, XXXIX, XL and XLI, and derivatives thereof, are either commercially available, are known in the literature, or may be obtained either by analogy with the processes described herein, or by conventional synthetic procedures, in accordance with standard techniques, from readily available starting materials using appropriate reagents and reaction conditions (e.g. as described hereinafter).
Substituents on the aromatic and/or non-aromatic, carbocyclic and/or heterocyclic ring(s) in compounds of formulae I, IV, V, VI, VII, VIII, IX, X, XI, XIII, XVI, XVIA, MX, XX, XII, XXII, XXIII, XXVI, XXIX, XXX, XXX, XXXII, XXXV, XXXV, XXXVI, XXXVII, XXXVIII, XXXX, XL and XLI may be introduced and/or interconverted using techniques well known to those skilled in the art. For example, nitro may be reduced to amino, hydroxy may be alkylated to give alkoxy, alkoxy may be hydrolysed to hydroxy, alkenes may be hydrogenated to alkanes, halo may be hydrogenated to H, etc.
The compounds of formula I may be isolated from their reaction mixtures using conventional techniques.
It will be appreciated by those skilled in the art that in the process described above the functional groups of intermediate compounds may need to be protected by protecting groups.
Functional groups which it is desirable to protect include hydroxy, amino and carboxylic acid. Suitable protecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl groups (e.g. t-butyldimethylsilyl, t-butyldiphenylsilyl or trimethylsilyl) and tetrahydropyranyl. Suitable protecting groups for carboxylic acid include C1-6 alkyl or benzyl esters. Suitable protecting groups for amino, amidino and guanidino include t-butyloxycarbonyl, benzyloxycarbonyl or 2-trimethylsilylethoxycarbonyl (Teoc). Amidino and guanidino nitrogens may also be protected by hydroxy or alkoxy groups, and may be either mono- or diprotected.
The protection and deprotection of functional groups may take place before or after coupling, or before or after any other reaction in the abovementioned schemes.
Protecting groups may be removed in accordance with techniques which are well known to those skilled in the art and as described hereinafter.
Persons skilled in the art will appreciate that, in order to obtain compounds of formula I in an alternative, and, on some occasions, more convenient, manner, the individual process steps mentioned hereinbefore may be performed in a different order, and/or the individual reactions may be performed at a different stage in the overall route (i.e. substituents may be added to and/or chemical transformations performed upon, different intermediates to those mentioned hereinbefore in conjunction with a particular reaction). This may negate, or render necessary, the need for protecting groups.
For example, this is particularly true in respect of the synthesis of compounds of formula I in which D1 or D2 (as appropriate) does not represent H, In this case, ORa and/or C(xe2x95x90X11)X12Rb groups may be introduced at an earlier stage in the overall synthesis using the process steps described hereinbefore.
Accordingly, the order and type of chemistry involved will dictate the need, and type, of protecting groups as well as the sequence for accomplishing the synthesis.
The use of protecting groups is fully described in xe2x80x9cProtective Groups in Organic Chemistryxe2x80x9d, edited by J W F McOmie, Plenum Press (1973), and xe2x80x9cProtective Groups in Organic Synthesisxe2x80x9d, 2nd edition, T W Greene and P G M Wutz, Wiley-Interscience (1991).
The protected derivatives of compounds of formula I may be converted chemically to compounds of formula I using standard deprotection techniques (e.g. hydrogenation). The skilled person will appreciate that certain compounds of the invention may be regarded as protected derivatives of other compounds of the invention.
Medical and Pharmaceutical Use
Compounds of the invention may possess pharmacological activity as such. Compounds of the invention that may possess such activity include, but are not limited to, those with a free amidine functionality as part of the structural fragment B.
However, other compounds of formula I (including those that do not possess such a free amidine functionality) may not possess such activity, but may be administered parenterally or orally, and thereafter metabolised in the body to form compounds that are pharmacologically active (including, but not limited to, corresponding free amidine compounds). Such compounds (which also include compounds that may possess some pharmacological activity, but that activity is appreciably lower than that of the active compounds to which they are metabolised to), may therefore be described as xe2x80x9cprodrugsxe2x80x9d of the active compounds.
Thus, the compounds of the invention are useful because they possess pharmacological activity, and/or are metabolised in the body following oral or parenteral administration to form compounds which possess pharmacological activity. The compounds of the invention are therefore indicated as pharmaceuticals.
According to a further aspect of the invention there is thus provided the compounds of the invention for use as pharmaceuticals.
In particular, the compounds of the invention are potent inhibitors of thrombin either as such and/or (e.g. in the case of prodrugs), are metabolised following administration to form potent inhibitors of thrombin, for example as demonstrated in the tests described below.
By xe2x80x9cprodrug of a thrombin inhibitorxe2x80x9d, we include compounds that form a thrombin inhibitor, in an experimentally-detectable amount, and within a predetermined time (e.g. about 1 hour), following oral or parenteral administration.
The compounds of the invention are thus expected to be useful in those conditions where inhibition of thrombin is required.
The compounds of the invention are thus indicated in the treatment and/or prophylaxis of thrombosis and hypercoagulability in blood and tissues of animals including man.
It is known that hypercoagulability may lead to thrombo-embolic diseases. Conditions associated with hypercoagulability and thrombo-embolic diseases which may be mentioned include inherited or acquired activated protein C resistance, such as the factor V-mutation (factor V Leiden), and inherited or acquired deficiencies in antithrombin III, protein C, protein S, heparin cofactor II. Other conditions known to be associated with hypercoagulability and thrombo-embolic disease include circulating antiphospholipid antibodies (Lupus anticoagulant), homocysteinemi, heparin induced thrombocytopenia and defects in fibrinolysis. The compounds of the invention are thus indicated both in the therapeutic and/or prophylactic treatment of these conditions.
The compounds of the invention are further indicated in the treatment of conditions where there is an undesirable excess of thrombin without signs of hypercoagulability, for example in neurodegenerative diseases such as Alzheimer""s disease.
Particular disease states which may be mentioned include the therapeutic and/or prophylactic treatment of venous thrombosis and pulmonary embolism, arterial thrombosis (e.g. in myocardial infarction, unstable angina, thrombosis-based stroke and peripheral arterial thrombosis) and systemic embolism usually from the atrium during arterial fibrillation or from the left ventricle after transmural myocardial infarction.
Moreover, the compounds of the invention are expected to have utility in prophylaxis of reocclusion (ie thrombosis) after thrombolysis, percutaneous trans-luminal angioplasty (PTA) and coronary bypass operations; the prevention of re-thrombosis after microsurgery and vascular surgery in general.
Further indications include the therapeutic and/or prophylactic treatment of disseminated intravascular coagulation caused by bacteria, multiple trauma, intoxication or any other mechanism; anticoagulant treatment when blood is in contact with foreign surfaces in the body such as vascular grafts, vascular stents, vascular catheters, mechanical and biological prosthetic valves or any other medical device; and anticoagulant treatment when blood is in contact with medical devices outside the body such as during cardiovascular surgery using a heart-lung machine or in haemodialysis.
In addition to its effects on the coagulation process, thrombin is known to activate a large number of cells (such as neutrophils, fibroblasts, endothelial cells and smooth muscle cells). Therefore, the compounds of the invention may also be useful for the therapeutic and/or prophylactic treatment of idiopathic and adult respiratory distress syndrome, pulmonary fibrosis following treatment with radiation or chemotherapy, septic shock, septicemia, inflammatory responses, which include, but are not limited to, edema, acute or chronic atherosclerosis such as coronary arterial disease, cerebral arterial disease, peripheral arterial disease, reperfusion damage, and restenosis after percutaneous trans-luminal angioplasty (PTA).
Compounds of the invention that inhibit trypsin and/or thrombin may also be useful in the treatment of pancreatitis.
According to a further aspect of the present invention, there is provided a method of treatment of a condition where inhibition of thrombin is required which method comprises administration of a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof, to a person suffering from, or susceptible to such a condition.
The compounds of the invention will normally be administered orally, intravenously, subcutaneously, buccally, rectally, dermally, nasally, tracheally, bronchially, by any other parenteral route or via inhalation, in the form of pharmaceutical preparations comprising active compound either as a free base, or a pharmaceutical acceptable non-toxic organic or inorganic acid addition salt, in a pharmaceutically acceptable dosage form. Depending upon the disorder and patient to be treated and the route of administration, the compositions may be administered at varying doses.
The compounds of the invention may also be combined and/or co-administered with any antithrombotic agent with a different mechanism of action, such as the antiplatelet agents acetylsalicylic acid, ticlopidine, clopidogrel, thromboxane receptor and/or synthetase inhibitors, fibrinogen receptor antagonists, prostacyclin mimetics and phosphodiesterase inhibitors and ADP-receptor (P2T) antagonists.
The compounds of the invention may further be combined and/or co-administered with thrombolytics such as tissue plasminogen activator (natural, recombinant or modified), streptokinase, urokinase, prourokinase, anisoylated plasminogen-streptokinase activator complex (APSAC), animal salivary gland plasminogen activators, and the like, in the treatment of thrombotic diseases, in particular myocardial infarction.
According to a further aspect of the invention there is thus provided a pharmaceutical formulation including a compound of the invention, in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier.
Suitable daily doses of the compounds of the invention in therapeutical treatment of humans are about 0.001-100 mg/kg body weight at peroral administration and 0.001-50 mg/kg body weight at parenteral administration.
The compounds of the invention have the advantage that they may be, or may be metabolised to compounds that may be, more efficacious, be less toxic, be longer acting, have a broader range of activity, be more potent, produce fewer side effects, be more easily absorbed than, or that they may have other useful pharmacological, physical, or chemical, properties over, compounds known in the prior art.
Biological Tests
Test A
Determination of Thrombin Clotting Time (TT)
The inhibitor solution (25 xcexcL) was incubated with plasma (25 xcexcL) for three minutes. Human thrombin (T 6769; Sigma Chem. Co) in buffer solution, pH 7.4 (25 xcexcL) was then added and the clotting time measured in an automatic device (KC 10; Amelung).
The clotting time in seconds was plotted against the inhibitor concentration, and the IC50TT was determined by interpolation.
IC50TT is the concentration of inhibitor in the test that doubles the thrombin clotting time for human plasma.
Test B
Determinaton of Thrombin Inhibition with a Chromogenic, Robotic Assay
The thrombin inhibitor potency was measured with a chromogenic substrate method, in a Plato 3300 robotic microplate processor (Rosys AG, CH-8634 Hombrechtikon, Switzerland), using 96-well, half volume microtitre plates (Costar, Cambridge, Mass., USA; Cat No 3690). Stock solutions of test substance in DMSO (72 xcexcL), 1 mmol/L, were diluted serially 1:3 (24+48 xcexcL) with DMSO to obtain ten different concentrations, which were analysed as samples in the assay. 2 xcexcL of test sample was diluted with 124 xcexcL assay buffer, 12 xcexcL of chromogenic substrate solution (S-2366, Chromogenix, Mxc3x6ndal, Sweden) in assay buffer and finally 12 xcexcL of a-thrombin solution, (Human xcex1-thrombin, Sigma Chemical Co.) both in assay buffer, were added, and the samples mixed. The final assay concentrations were: test substance 0.00068-13.3 xcexcmol/L, S-2366 0.30 mmol/L, xcex1-thrombin 0.020 NIHU/mL. The linear absorbance increment during 40 minutes incubation at 37xc2x0 C. was used for calculation of percentage inhibition for the test samples, as compared to blanks without inhibitor. The IC50-robotic value, corresponding to the inhibitor concentration which caused 50% inhibition of the thrombin activity, was calculated from a log concentration vs. % inhibition curve.
Test C
Determinaton of the Inhibition Constant Ki for Human Thrombin
Ki-determinations were made using a chromogenic substrate method, performed at 37xc2x0 C. on a Cobas Bio centrifugal analyser (Roche, Basel, Switzerland). Residual enzyme activity after incubation of human xcex1-thrombin with various concentrations of test compound was determined at three different substrate concentrations, and was measured as the change in optical absorbance at 405 nm.
Test compound solutions (100 xcexcL; normally in buffer or saline containing BSA 10 g/L) were mixed with 200 xcexcL of human xcex1-thrombin (Sigma Chemical Co) in assay buffer (0.05 mol/L Tris-HCl pH 7.4, ionic strength 0.15 adjusted with NaCl) containing BSA (10 g/L), and analysed as samples in the Cobas Bio. A 60 xcexcL sample, together with 20 xcexcL of water, was added to 320 xcexcL of the substrate S-2238 (Chromogenix AB, Mxc3x6lndal, Sweden) in assay buffer, and the absorbance change (xcex94A/min) was monitored. The final concentrations of S-2238 were 16, 24 and 50 /xcexcmol/L and of thrombin 0.125 NIH U/mL.
The steady state reaction rate was used to construct Dixon plots, i.e. diagrams of inhibitor concentration vs. 1/(xcex94A/min). For reversible, competitive inhibitors, the data points for the different substrate concentrations typically form straight lines which intercept at x=xe2x88x92Ki.
Test D
Determination of Activated Partial Thromboplastin Time (APTT)
APTT was determined in pooled normal human citrated plasma with the reagent PTT Automated 5 manufactured by Stago. The inhibitors were added to the plasma (10 xcexcL inhibitor solution to 90 xcexcL plasma) and incubated with the APTT reagent for 3 minutes followed by the addition of 100 xcexcL of calcium chloride solution (0.025M) and APTT was determined in the mixture by use of the coagulation analyser KC10 (Amelung) according to the instructions of the reagent producer. The clotting time in seconds was plotted against the inhibitor concentration in plasma and the IC50APTT was determined by interpolation.
IC50APTT is defined as the concentration of inhibitor in human plasma that doubled the Activated Partial Thromboplastin Time.
Test E
Determination of Thrombin Time ex vivo
The inhibition of thrombin after oral or parenteral administration of the compounds of formula I, dissolved in ethanol:Solutol(trademark):water (5:5:90), were examined in conscious rats which, one or two days prior to the experiment, were equipped with a catheter for blood sampling from the carotid artery. On the experimental day blood samples were withdrawn at fixed times after the administration of the compound into plastic tubes containing 1 part sodium citrate solution (0.13 mol per L) and 9 parts of blood. The tubes were centrifuged to obtain platelet poor plasma. The plasma was used for determination of thrombin time as described below.
The citrated rat plasma, 100 xcexcL, was diluted with a saline solution, 0.9%, 100 xcexcL, and plasma coagulation was started by the addition of human thrombin (T 6769, Sigma Chem Co, USA) in a buffer solution, pH 7.4, 100 xcexcL. The clotting time was measured in an automatic device (KC 10, Amelumg, Germany).
Where a xe2x80x9cprodrugxe2x80x9d compound of formula I was administered, concentrations of the appropriate active thrombin inhibitor of formula I (e.g. the free amidine or guanidine compound) in the rat plasma were estimated by the use of standard curves relating the thrombin time in the pooled citrated rat plasma to known concentrations of the corresponding xe2x80x9cactivexe2x80x9d thrombin inhibitor dissolved in saline.
Based on the estimated plasma concentrations of the active thrombin inhibitor (which assumes that thrombin time prolongation is caused by the aforementioned compound) in the rat, the area under the curve after oral and/or parenteral administration of the corresponding prodrug compound of formula I was calculated (AUCpd) using the trapezoidal rule and extrapolation of data to infinity.
The bioavailability of the active thrombin inhibitor after oral or parenteral administration of the prodrug was calculated as below:
[(AUCpd/dose)/(AUCactive,parenteral/dose]xc3x97100
where AUCactive,parenteral represents the AUC obtained after parenteral administration of the corresponding active thrombin inhibitor to conscious rats as described above.
Test F
Determination of Thrombin Time in Urine ex vivo
The amount of the xe2x80x9cactivexe2x80x9d thrombin inhibitor that was excreted in urine after oral or parenteral administration of xe2x80x9cprodrugxe2x80x9d compounds of the invention, dissolved in ethanol:Solutol(trademark):water (5:5:90), was estimated by determination of the thrombin time in urine ex vivo (assuming that thrombin time prolongation is caused by the aforementioned compound).
Conscious rats were placed in metabolism cages, allowing separate collection of urine and faeces, for 24 hours following oral administration of compounds of the invention. The thrombin time was determined on the collected urine as described below.
Pooled normal citrated human plasma (100 xcexcL) was incubated with the concentrated rat urine, or saline dilutions thereof, for one minute. Plasma coagulation was then initiated by the administration of human thrombin (T 6769, Sigma Chem Company) in buffer solution (pH 7.4; 100 xcexcL). The clotting time was measured in an automatic device (KC 10; Amelung).
The concentrations of the active thrombin inhibitor in the rat urine were estimated by the use of standard curves relating the thrombin time in the pooled normal citrated human plasma to known concentrations of the aforementioned active thrombin inhibitor dissolved in concentrated rat urine (or saline dilutions thereof). By multiplying the total rat urine production over the 24 hour period with the estimated mean concentration of the aforementioned active inhibitor in the urine, the amount of the active inhibitor excreted in the urine (AMOUNTpd) could be calculated.
The bioavailability of the active thrombin inhibitor after oral or parenteral administration of the prodrug was calculated as below:
[(AMOUNTpd/dose)/(AMOUNTactive,parenteral/dose]xc3x97100
where AMOUNTactive,parenteral represents the amount excreted in the urine after parenteral administration of the corresponding active thrombin inhibitor to conscious rats as described above.
Test G
Metabolic Activation of Prodrug Compounds in vitro
Prodrug compounds of formula I were incubated at 37xc2x0 C. with liver microsomes or 10 000 g (referring to the centrifuge speed) supernatant fractions (i.e. s9 fraction) prepared from human or rat liver homogenate. The total protein concentration in the incubations were 1 or 3 mg/mL dissolved in 0.05 mol/L TRIS buffer (pH 7.4), and with the cofactors NADH (2.5 mmol/L) and NADPH (0.8 mmol/L) present. The total volume of the incubate was 1.2 mL. The initial prodrug concentrations were 5 or 10 xcexcmol/L. Samples were collected from the incubate at regular intervals more than 60 minutes after the start of the incubations. Samples (25 xcexcL) from the incubate were mixed with an equal volume of human or rat plasma and an appropriate amount of thrombin, and the clotting time (i.e. thrombin time) was measured on a coagulometer (KC 10; Amelumg). The amount of xe2x80x9cactivexe2x80x9d thrombin inhibitor formed was estimated by the use of standard curves relating the thrombin time in pooled citrated human or rat plasma to known concentrations of the corresponding xe2x80x9cactive thrombin inhibitorxe2x80x9d.